Cockroach myotropic peptides

The octapeptide leucopyrokinin (LPK) was the eleventh and final myotropic peptide isolated from L. maderae (11). Unlike the leucokinins and LSK s, LPK exhibits a myotropic activity on the muscles of the cockroach foregut and oviduct (Holman, G. M. and Nachman, R. J., unpublished observation.). LPK was the highest titered of the Leucophaea myotropins (1.36 pmol/head) but was the least potent on the isolated hindgut preparation (threshold concentration -0.6 nM). LPK shares a 50% sequence identity with Pea-HTH-II at the 1, 4, 5, and 6 positions and is amidated at the C-terminus. Yet LPK does not contain Trp, which is present at position 8 in every AKH/RPCH peptide. Furthermore, LPK is positively charged with a residue of Arg at position 8. These structural observations show that LPK is not a member of the AKH/RPCH family. A study of LPK analogs supports this view (20). 

Holman, G M., Nachman, R J, and Wright, M S (1990) A strategy for the isolation and structural characterization of certain insect myotropic peptides that modify the spontaneous contractions of the isolated cockroach hindgut, in Chromatography and Isolation of Insect Hormones and Pheromones (MacCaffery, A R and Wilson, I. D., eds), Plenum, New York, pp 195-204. 

It was determined that the minimal peptide sequence required to stimulate pheromone biosynthesis was the C-terminal 5 amino acids, FXPRLamide, and that the carboxy terminus needs to be amidated [148,149]. This sequence was also established as the minimal sequence required for myotropic activity in cockroaches [ 150] and induction of embryonic diapause in B. mori [ 151 ]. Crossreactivity of peptides containing the FXPRLamide motif was also established for myotropic, diapause induction, and pheromone biosynthesis [152-154]. Therefore, the common C-terminal FXPRLamide defines this family of peptides. A partial listing of peptides identified to date is shown in Table 1. 

The 1-125 protein column (operated in a normal-phase mode) was chosen as the final purification step for two reasons 1) the small (sometimes invisible) amount of residue remaining after the previous purification steps was easily dissolved in 95% acetonitrile 5% water (the initial-conditions solvent for this system) and 2) the peaks collected from this column were in a solution (75%-85% acetonitrile) suitable for peptide stabilization and required no further manipulation (other than capping) prior to storage. The structures of the 11 myotropic cockroach peptides isolated with this method are listed, along with appropriate references, in Table I (leucokinins... 

The leucokinins (LK s) are a new class of insect myotropic neuropeptides isolated from head extracts of the cockroach L. maderae. These octapeptides all contain a similar core sequence of 5 amino acids that extend from position 4 through 8. This sequence Phe-X-Ser-Trp-Gly-NH2 seems to be required for hindgut stimulation. The initial response of the hindgut to the LK s was characterized by an increase in the frequency and/or amplitude of phasic contractions (50, ). At higher peptide concentrations, a tonic component was generally present. All of the LK s showed a response at 3 x 10 M that was 5-10% above the mean level of spontaneous activity. The maximum response for each of the peptides was recorded at a concentration 2.1 X 10" M. Thus, the intrinsic activities for the LK s are nearly equal because the dose-response curves have about the same asymptotic limits. A comparison of the dose concentrations that gave a half maximal response (ED q) for the 8 peptides is shown in Table I. 

The related achetakinins double the rate of fluid secretion by isolated Acheta Malpighian tubules (Coast, G. M., et al. J. Insect Phvsiol.. in press.)- In addition, some leucokinins stimulate fluid secretion and/or depolarize transepithelial membrane potentials in the malpighian tubules of the yellow fever mosquito, Aedes aegvpti. in a fashion similar to several uncharacterized peptides isolated from mosquito head extracts (23). Thus, the neuropeptide family of leucokinins, achetakinins, and homologs may function in the control of water and ion balance, as well as myotropic activity, in a number of insects. To determine the relationships of the C-terminal sequence of the leucokinins to myotropic activity, analogs were synthesized and tested on the isolated cockroach hindgut. 

Locustakinin, H-Ala-Phe-Ser-Ser-Trp-Gly-NH2, a 6-peptide amide from Locusta mi-gratoria. Locustakinin is a myotropic neuropeptide with sequence similarity to cockroach leucokinins [L. Schoofs et al., Regul. Pept. 1992, 37, 49 G. M. Coast, Peptides 1996, 17, 327]. 

Only the C-terminal octapeptide fragment is required to elicit a full myostimulatory response on the isolated cockroach hindgut, indeed this fragment is seven-fold more potent than the parent undecapeptide leucosulfakinin (LSK) peptide. The C-terminal hexapeptide, which contains the critical sulfate moiety, is the active core sequence for myotropic activity (17). The sulfakinin receptor exhibits some tolerance to movement of the position of the Tyr-sulfate group in interactions with sulfakinin peptide analogs, particularly to a shift of one position towards the N-terminus (18). 

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